Extracellular ATP promotes angiogenesis and adhesion of TNBC cells to endothelial cells via upregulation of CTGF.

Our previous works have indicated that extracellular ATP is an important prometastasis factor. However, the molecular mechanism involved needs to be further studied. We demonstrated that extracellular ATP treatment could upregulate the expression of connective tissue growth factor (CTGF) in both triple-negative breast cancer (TNBC) cells and endothelial cells (ECs). Extracellular ATP stimulated the migration of TNBC cells and ECs, and angiogenesis of ECs via the P2Y2--YAP-CTGF axis. Furthermore, we demonstrated that adenosine triphosphate (ATP) stimulated TNBC cell adhesion to ECs and transmigration through the EC layer via CTGF by upregulation of integrin ß1 on TNBC cells and VCAM-1 on ECs. Both apyrase (ATP-diphosphohydrolase) and CTGF shRNA treatments could inhibit the metastasis of inoculated tumors to lung and liver in a mouse model, and these treated tumors had fewer blood vessels. Collectively, our data indicated that extracellular ATP promotes tumor angiogenesis and the interactions between TNBC cells and ECs through upregulation of CTGF, thereby stimulating TNBC metastasis. The pleiotropic effects of ATP in angiogenesis and cell adhesion suggest that extracellular ATP or CTGF could be an effective target for TNBC therapy.

Extracellular ATP promotes breast cancer invasion and epithelial-mesenchymal transition via hypoxia-inducible factor 2α signaling.

Extracellular ATP has been shown to play an important role in invasion and the epithelial-mesenchymal transition (EMT) process in breast cancer; however, the mechanism is unclear. Here, by using a cDNA microarray, we demonstrated that extracellular ATP could stimulate hypoxia-inducible factor (HIF) signaling and upregulate hypoxia-inducible factor 1/2α (HIF-1/2α) expression. After knocking down HIF-1/2α using siRNA, we found that ATP-driven invasion and EMT were significantly attenuated via HIF2A-siRNA in breast cancer cells. By using ChIP assays, we revealed that the biological function of extracellular ATP in invasion and EMT process depended on HIF-2α direct targets, among which lysyl oxidase-like 2 (LOXL2) and matrix metalloproteinase-9 (MMP-9) mediated ATP-driven invasion, and E-cadherin and Snail mediated ATP-driven EMT, respectively. In addition, using silver staining and mass spectrometry, we found that phosphoglycerate kinase 1 (PGK1) could interact with HIF-2α and mediate ATP-driven HIF-2α upregulation. Furthermore, we demonstrated that expressions of HIF-2α and its target proteins could be regulated via ATP by AKT-PGK1 pathway. Using a Balb/c mice model, we illustrated the function of HIF-2α in promoting tumor growth and metastasis in vivo. Moreover, by exploring online databases, we found that molecules involved in ATP-HIF-2α signaling were highly expressed in human breast carcinoma tissues and were associated with poor prognosis. Altogether, these findings suggest that extracellular ATP could promote breast carcinoma invasion and EMT via HIF-2α signaling, which may be a potential target for future anti-metastasis therapy.

The risk variant rs11836367 contributes to breast cancer onset and metastasis by attenuating Wnt signaling via regulating NTN4 expression.

Most genome-wide association study (GWAS)-identified breast cancer-associated causal variants remain uncharacterized. To provide a framework of understanding GWAS-identified variants to function, we performed a comprehensive study of noncoding regulatory variants at the NTN4 locus (12q22) and NTN4 gene in breast cancer etiology. We find that rs11836367 is the more likely causal variant, disrupting enhancer activity in both enhancer reporter assays and endogenous genome editing experiments. The protective T allele of rs11837367 increases the binding of GATA3 to the distal enhancer and up-regulates NTN4 expression. In addition, we demonstrate that loss of NTN4 gene in mice leads to tumor earlier onset, progression, and metastasis. We discover that NTN4, as a tumor suppressor, can attenuate the Wnt signaling pathway by directly binding to Wnt ligands. Our findings bridge the gaps among breast cancer-associated single-nucleotide polymorphisms, transcriptional regulation of NTN4, and breast cancer biology, which provides previously unidentified insights into breast cancer prediction and prevention.